Structure of the chromatin remodelling enzyme Chd1 bound to a ubiquitinylated nucleosome

Ramasubramanian Sundaramoorthy; Amanda L. Hughes; Hassane El-Mkami; David Norman; Tom Owen-Hughes

The movie frames were subjected to frame wise motion correction using MotionCor2. CTF correction was then performed on the motion corrected summed image using Gctf. Subsequent image processing was performed with RELION 2.0.4. About 5000 particles from 50 micrographs were first handpicked in RELION, extracted and 2D classes were generated. These 2D classes were then used as a reference in RELION autopick routine and particles were picked from respective number of micrographs from each dataset. The autopicked particles were subsequently extracted and sorted. An iterative round of two-dimensional classification was performed to discard poorly averaging particles, contamination and exploded particles. On the resultant cleaned up particle stack a hierarchical three-dimensional classification and refinement was performed as described in the results. A low pass filtered low resolution chd1 engaged nucleosome structure was used as an initial model in the 3D classification. At the three-dimensional refinement stages a mask that encompasses the entire Chd1-nucleosome complex was applied. Post processing of refined models was performed with automatic B factor determination in RELION. Local resolution estimates were determined using Resmap-1.4.